A common result of prolonged alcohol consumption is liver damage. There is a considerable body of evidence that implicates the first metabolite of ethanol oxidation, acetaldehyde (AA), and malondialdehyde (MDA), a by-product of lipid peroxidation, as the precursors to hepatotoxicity. When proteins are incubated with MDA and AA, hybrid protein adducts, designated as MAA adducts, are generated. To date we have determined the structures of two MAA adducts. One adduct is the fluorescent 1,4-dihydopyridine 3,5-dicarboxaldehyde, formed by a Hantzsch reaction. The other is the nonfluorescent 2-formyl-3-alkylamino-butanal, which results from a Mannich reaction. Both adducts can form on a protein via the t-amino group of lysine or the N-terminus whereas the Mannich adduct can also form on a histidine residue. The research proposed here will focus on identifying the specific sites of MAA modifications on proteins. First, each adduct will be synthesized on model peptides and their mass spectra studied to determine the characteristic fragmentation patterns of each adduct. Next we will MAA-modify human alpha-calcitonin gene-related peptide, a 37 residue peptide that has 2 lysines and 1 histidine, and use mass spectrometry to identify the sites of MAA adduct formation. Finally, the proteins ribonuclease A (RNase A) and soybean trypsin inhibitor (STI) will be used in similar experiments. Both proteins have been extensively studied and their structures are well characterized. In addition, each protein has only 10 lysine residues and thus, mass spectrometry should be able to determine the sites of adduct formation.